The regulation of transcription is a key step in many biological processes in eukaryotic cells, including control of cell growth and division, differentiation and development of tissues and organs, and response to extracellular signals and environmental conditions. The long-term objective of this project is an understanding of the molecular mechanisms of transcriptional activation. Lytic infection by herpes simplex virus represents a useful model system for this objective, in that a component of the HSV virion (termed VP16) specifically and potently activates of the viral immediate-early genes. The activation domain of VP16 is among the most potent known to date, and thus has been widely adopted as a paradigm for the study of this phenomenon. The specific aims for this project period will employ a broad range of biochemical and molecular genetic methods to explore the critical structural features of the VP16 transcriptional activation domain. Oligonucleotide-directed mutagenesis will be used to test specific hypotheses emerging from previous work regarding the roles of particular amino acids or structural motifs. To identify features critical to VP16 function that might not be targeted by current hypotheses, randomly- generated mutants will be identified through in vivo selection and screening assays in yeast. Physical descriptions of the secondary and tertiary structures of VP16 will be sought using differential scanning calorimetry, Fourier-transform infrared spectroscopy, circular dichroism spectroscopy, two-dimensional nuclear magnetic resonance, and crystallographic techniques. Finally, identification and characterization of VP16 homologs in related viruses of economic and agricultural importance will be pursued to reveal structural features conserved through the natural selection events undergone by these viruses. This project will not only add significantly to an understanding of the mechanisms of transcriptional activation, but will also provide knowledge necessary to explore antiviral strategies for human and veterinary use and for exploitation of HSV vectors as agents for selective gene therapy.